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Purification and characterization of acute phase rat plasma thiostatin
M E Rusiniak1, P V Wagh, G S Bedi
1Department of Biochemical Pharmacology, State University of New York at Buffalo, New York 14260.
Thiostatin was purified from acute phase plasma of turpentine-treated rats by a novel, single-step carboxymethyl-papain Sepharose 4B column chromatographic procedure. Purified thiostatin appeared as a single band in SDS-PAGE with an estimated molecular weight of 68,000. Western blot with polyclonal rabbit anti-thiostatin IgG confirmed a homogeneous immuno-reactive 68 kDa species. Specific activity, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.972 mg kininogen equivalent per mg protein. The yield of thiostatin exceeded 60% and the protein was purified 10.7-fold.
Thiostatin was purified from acute phase plasma of turpentine-treated rats by a novel, single-step carboxymethyl-papain Sepharose 4B column chromatographic procedure. Purified thiostatin appeared as a single band in SDS-PAGE with an estimated molecular weight of 68,000. Western blot with polyclonal rabbit anti-thiostatin IgG confirmed a homogeneous immuno-reactive 68 kDa species. Specific activity, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.972 mg kininogen equivalent per mg protein. The yield of thiostatin exceeded 60% and the protein was purified 10.7-fold.